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M.r. Eslaminejad, L. Taghiyar, S. Kiani, A. Piryaee,
Volume 4, Issue 2 (8-2007)
Abstract
Abstract Background and Objectives Mesenchymal stem cells are appropriate candidates to treat diseases including articular cartilage defects. There are plenty of researches being conducted to make the application of these cells possible. The purpose of this study was to cultivate murine mesenchymal stem cells (MSCs) in alginate gel and transplant them subcutaneously to immuno-suppressed rats to examine their chondrogenic potential in vivo. Materials and Methods 4-6 week old NMRI mice were sacrificed and their bone marrow cells were cultivated in 6-cell plates at the density of 500 cell/cm2. The pure fibroblastic cells appeared after two passages. 2×106 fibroblastic cells were mixed with 1 ml of alginate solution and converted into gel by being exposed to calcium chloride solution. MSCs-embedded alginate gel were then transplanted subcutaneously to 6 rats that had received an immunosuppressive drug (cyclosporine) for transplant rejection to be avoided. 5 weeks after transplantation, the alginate gels were removed and evaluated by histochemistry, RT-PCR for certain cartilage markers, and transmission electron microscopy. Results 5 weeks after transplantation, the skin was incised and the alginate gel with its surrounding vascular connective tissue were removed. Tuloidine blue staining indicates that the cells within the gel assumed oval morphology and occupied lacuna-like cavities. RT-PCR analysis revealed that in these cells the mRNA of some cartilage markers such as collagen II (the marker of hyaline cartilage), collagen X (hypertrophied chondrocyte marker in osteogenesis), and aggreacan were largely produced. Ultra-thin sections analysis showed that the cells within the lacuna-like cavity of alginate gel contain a large amount of expanded rough endoplasmic reticulum and secret fibrillar extra cellular matrix. Conclusions Transplanted murine MSCs cultivated in alginate gel can differentiate into hyaline cartilage with the sign of osteogenic initiation. Key words: Mesenchymal stem cells, Alginate, Transplantation
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Volume 4, Issue 2 (8-2007)
Abstract
Abstract Background and Objectives The isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures are arduous due to the unwanted growth of hematopoietic cells and non-MSCs. In this study, homogenous populations of CD34+ MSCs from mouse bone marrow were purified through positive immunoselection. Materials and Methods In this experimental study, C57BL/6 mice were sacrificed. Their bone marrow cells were aspirated and incubated with anti-CD34 conjugated magnetic beads. After immunoselection, a sample of the cells was prepared for flow cytometry in order to examine the expression of CD34 antigen and the remains were cultivated. 24 hours later, non-adherent cells, mostly consisting of CD34+ hematopoietic stem cells were removed and the plastic adherent cultivated cells were investigated for surface markers (CD44, Sca-1, Vcam-1, CD34, CD11b and CD45). The plastic adherent cultivated cells were differentiated into the osteoblastic and adipogenic lineages to investigate the mesenchymal nature. Furthermore, the expression of some surface markers were investigated through flow cytometry. Results Purified populations of fibroblast-like CD34+ cells were achieved in the first passage (one week after initiative cultivation). The CD34, CD44, Sca-1 and Vcam-1 markers were expressed but CD11b and CD45 were absent. Osteocyte (osteocalsin, osteopontin, parathyroid hormone receptor) and adipocyte (lipo protein lipase and adipsin) differentiating genes were expressed in these cells. � Conclusions This study indicates that this protocol can result in efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We showed that plastic adherent murine bone marrow derived CD34+ cells with capability of differentiating into skeletal lineages in vitro are MSCs. � Key words: Mesenchymal stem cells, Bone marrow, CD34 antigen
M. Kheirandish, M. Ebtekar, A.a. Pourfathollah, Z. Mohammad Hassan, S.d. Siadat, A. Kazem Nejad,
Volume 4, Issue 3 (9-2007)
Abstract
Abstract Background and Objectives The incidence and severity of Graft Versus Host Disease following the use of umbilical cord blood as a source of stem cells for bone marrow reconstitution challenge the scientific findings of the immunocompetence of newborn immune cells. The reports show that self renewal characteristics and the proliferative capacity of primitive hematopoietic progenitors in the preterm cord blood are higher in comparison with term cord blood and bone marrow. In this study, the characteristics of preterm cord blood immune cells were analyzed from a naive point of view especially in comparison with its counterparts in term cord blood. Materials and Methods Term and pretem MNCs were isolated and cultivated in complete media containing PMA (50 ng/ml) and Ionomycine (1µg/ml) at the presence of monensin they were then permeabilized with %0.1 saponin. After cell stimulation with PMA and Ionomycine, staining was performed with Moab anti-CD69 antibody to estimate the level of activation and was also conjugated with anti- IL-10, IFN-γ, IL-4, IL-2 antibody to evaluate the production of cytokine. Mean percentage frequency of cytokine producing cells and the level of cytokine expression in CD4+/CD8+, CD45RA+/RO+ cells were analyzed by Epics-XL and IMMUNO-4 software. Statistical analysis was carried out using Kolmogrov-Smirnov and Student's t-test . Results Cellular phenotypic analysis showed no significant differences in CD4 + CD45RA+, CD8+CD45RO+ and CD4+CD45RO+ cells in term and preterm cord blood (p< 0.05). Mean percentage of CD3+ , CD4+ , CD8+, HLA-DR+CD3+, DR - HLA+CD4+ and CD25+ cells had significant increment in term cord blood. No statistically significant differences in the level of expression and the frequency of cytokine producing cells were observed in distinct gestational ages. Conclusions Considering the lack of any significant functional differences between term and preterm cord blood immune cells, hematopoietic stem cells of preterm cord blood not only have the same immunological behavior, especially with respect to GVHD, but also have the higher frequency and proliferative capacity. The presence of immature progenitor cells may have priority to term cord blood and be applied in transplantation settings. � Key words:�Cord blood, CD4 positive T lymphocyte , CD8 positive T lymphocyte, Cytokine
A. Jafari Kermani, F. Fathi, S.j. Mowla, Y. Gheisari,
Volume 4, Issue 4 (2-2008)
Abstract
Abstract Background and Objectives Stem cells are defined with two main characteristics which are self renewal potential and pluripotency. It is of great importance to study expression of self renewal genes in different stem cells, because self renewal regulatory mechanisms may vary among different kinds of stem cell. Human umbilical cord stem cells have recently gained so much attention, because their use in cell therapy methods has several advantages. The main objective of the present study was to evaluate the expression of Oct-4, Sox-2, Nanog, Nucleostemin, Bmi-1 and Sox-9 self renewal genes in human umbilical cord vein mesenchymal stem cells. Materials and Methods In this experimental study, umbilical cords were obtained in sterile condition and carried to the laboratory in HBSS buffer. Mesenchymal stem cells were harvested by collagenase treatment and cultured by means of their adhesiveness to plastic surfaces in DMEM medium supplemented with 15% FBS. To confirm the mesenchymal identity of these cells, expression of some surface markers including CD105, CD106, CD54, CD45, HLA-DR, CD166, and CD34 was analysed by means of flowcytometery. Finally, expression of self renewal genes was evaluated by RT-PCR and immunocytochemistry techniques. Results Flowcytometry analysis of UBMCs revealed that the cells are positive for CD105 (98%) and negative for CD34 (0%), CD54 (1%), CD45 (0%), CD106 (0%), CD166 (0%), and HLA-DR (0%). Our results also revealed Oct-4, Nanog, Nucleostemin, Bmi-1 and Sox-9 expression in human umbilical cord vein mesenchymal stem cells, but not Sox-2 self renewal gene expression. Conclusions The results showed that the mechanism which is known today for Oct-4, Nanog and Sox-2 function is not the only possible mechanism in which these three genes can play role to regulate self renewal potential of stem cells. Obtained results can offer that a combination of regulatory mechanisms which regulate self renewal of adult and embryonic stem cells, may be responsible to regulate self renewal of stem cells. � Key words : Mesenchymal stem cells, Umbilical cord, PCR, Immunocytochemistry
Dr. M.r. Eslaminejad, L. Rouhi, Dr. S.m. Arab Najafi, Dr. H. Baharvand,
Volume 5, Issue 1 (3-2008)
Abstract
Abstract
Background and Objectives
In current protocol for isolation and expansion of mesenchymal stem cells (MSCs), the use of fetal calf serum (FBS) as a medium supplement is inevitable. FBS is immunogenic for human subjects and may transfer infection in the case of transplantation. In the search for an appropriate substitute for FBS, in the present study the effect of plasma prepared from peripheral blood on the growth of MSCs has been examined.
Materials and Methods
Marrow cells obtained from rat long bones were cultivated both in the mediums containing FBS and in those with plasma prepared from rat peripheral blood for the primary culture and the three consequent passages. The cells present in all four passages were evaluated at each culture stage for population doubling number, viability, and the rate of proliferation by cell count, MTT assay, and plotting growth curve, respectively. The cells from primary culture were also examined with respect to their clonogenic activity. All experiments were replicated 10 times and the average values for each group were statistically compared. Furthermore, passage 3 cells from each group were examined in terms of bone and adipogenic differentiation by specific staining as well as RT-PCR.
Results
The cultures from plasma group appeared morphologically more homogenous than those from FBS group. In general, the cells from FBS groups had a better status in terms of total population doubling number and MTT test but these differences were not significant in passage 3. Moreover, growth curve plotted for each group indicated that the proliferation of cells in the plasma group is somewhat slower than those in FBS groups. The culture of plasma groups showed more colon cells compared to FBS groups but the colons in the latter appeared larger than the former. The cells from both groups were readily differentiated into osteoblastic and adipogenic cells lineages; this was confirmed by alizarin red and oil red staining, the bone expression of osteopontin and osteocalcin, and the expression of PPAR-alfa, PPAR-gamma, and C/EBP-alpha for adipose cells.
Conclusions
Taken together, plasma as a substitute for FBS can support proliferation of MSCs and maintains their viability in vitro. Although this support was somehow less strong than FBS, plasma could still be considered a safe substitute for FBS.
M. Ghorbani, M. Soleimani, S. Kavyani, A. Atashi,
Volume 5, Issue 2 (8-2008)
Abstract
Abstract Background and Objectives Embryonic stem cells are potential pluripotent stem cells derived from inner cell mass of embryonic blastocyst stage. So far, differentiation of embryonic stem cells to megakaryocyte lineage has resulted from coculture with bone marrow stromal cells with disadvantages such as possibility of stem cell contamination from feeder layer and secretion of unknown factors from this layer ending in unwanted differentiation and genetic changes in embryonic stem cells. In this study, while excluding feeder layer, we differentiated murine bone marrow cells to megakaryocyte lineage. Materials and Methods In this experimental study, embryoid bodies resulted from R1 murine embryonic stem cells. The embryoid bodies were then cultured in two groups of test (containing IMDM with FBS, L-glutamin, non-essential aminoacids, beta mercaptoethanol, and TPO and IL-3 as growth factors) and control (the same culture medium used in test group without growth factors). After four days the colony assay test was performed, and after eight days immunohistochemistry staining for CD41 molecule, and finaly transcription test for PF4 gene using RT-PCR to demonstrate differentiation to megakaryocyte lineage. Results Results of the colony assay test indicated that the cells considered had the capacity to give rise to colonies. The total number of colonies and banzidine positive colonies were 57 ± 6.1 and 18 ± 3.6 respectively. On the other hand, immunocytochemistry analysis and PCR showed that the CD41 molecule and PF4 gene are expressed in differentiated cells. Conclusions Our results indicated that murine embryonic stem cells differentiate into megakaryocyte lineage cells without utilizing feeder layer, and embryonic stem cells can be used as an immortal new source for producing megakaryocyte lineage cells on which basic biological research can be conducted. � Key words : Embryonic stem cells, Megakaryocytic, Cell differentiation
Dr. M. Shaiegan, S. Mohammadi, Sh. Samiee, Dr. K. Alimoghadam, Dr. Gh. Babaei, A. Ghashghaiee, Sh. Rostami, Dr. F. Kkatami, Dr. A. Azarkeivan, Dr. S. Zolfaghari, Z. Ataiee, Dr. A. Ghavamzadeh,
Volume 6, Issue 1 (5-2009)
Abstract
Abstract Background and Objectives It is suggested that HA-1 mismatching among hematopoietic stem cell recipients-donors be associated with acute graft-versus-host disease (aGVHD). So the aim of this study was to evaluate HA-1 frequency and examine the correlation between HA-1 disparity and GVHD patients who received transplantation from HLA-A2 identical siblings. Materials and Methods Extracted DNA samples were collected from 55 HLA-A2-positive donor-recipient pairs. All the patients received peripheral blood stem cell transplant (PSCT) from HLA-identical siblings. HA-1 was detected by SSP-PCR method. The HA-1 typing was performed using SSP method. Data were analyzed using Chi-square, Man withney and Z test with SPSS 11.5. Results Thirty patients showed to be GVHD I-IV and 25 pairs were without any GVHD signs. The frequency rates of HA-1R and HA-1H alleles in patients were 0.55 and 0.45, respectively it showed no significant difference with the frequency rates (0.53 and 0.47) of this allels in donors (p>0.05). HA-1 disparity was detected in 8 out of the 55 donor/recipient pairs (14.5%). aGVHD ( grades I-IV ) was occurred in 6 of patients. Two patients with HA-1 disparity did not show any GVHD signs. X2 test showed there was not any relationship between the incidence of acute graft-versus-host-disease (aGVHD) and HA-1 incompatibility in the patiens. Conclusions In spite of higher frequency of HA-1 disparity in GVHD+ group, our data did not reflect any significant association between HA-1 disparity and risk of acute GVHD. Key words : Polymerase Chain Reaction, Graft- Versus – Host Disease, Minor Histocompatibility Antigens , Hematopoietic Stem Cell Transplantation
Dr. M. Khani, Dr. K. Alimoghadam, Dr. A. Karimi, A. Mousavi, Dr. A. Ghavamzadeh,
Volume 6, Issue 1 (5-2009)
Abstract
Abstract Background and Objectives Multiple myeloma (MM) is a clonal disorder of hematopoietic stem cell. We have been doing in-patient stem cell transplant (SCT) in Iran since 1991 however, this is the first time we have decided to embark on out-patient SCT. Materials and Methods In this cohort study, the selected Multiple Myeloma patients received outpatient stem cell transplantation. They were then discharged and followed by an out-patient SCT team including a general physician, a staff nurse and a care giver during the neutropenic period. All data were collected and analyzed using ANOVA test, Caplan mayer curve and SPSS 11.5. Results Forty four patients received in-patient or out-patient autologus SCT. The rates of median hospital stay were 28 (19-54) and 6.5 days (1-8) in in-patient and out-patient groups, respectively. Median home visit by team was 10.5 days. There were not significant differences (p<0.001) between these groups as far as apheresis days, granulocyte colony stimulatig factor (GCSF) dosage as mobilization, number of mono nuclear cell (MNC) or cluster of differentiation (CD)34+ cell parameters are concerned. There was also a significant decrease in total cost of SCT in out-patient group by 70% (p<0.017) including visit cost with 80% decrease (p<0.01), drug cost with 50% (p<0.004), laboratory cost with 70% (p<0.02), and hospital cost with 70% (p<0.04). Conclusions Our results show that out-patient autolgous SCT in multiple myeloma patients is feasible and its complications are manageable. Significant reduction in cost and bed requirement is also inevitable. Key words : Multiple Myeloma, Stem Cell Transplantation, Apheresis
B. Beiki, Dr. M. Ebrahimi,
Volume 6, Issue 2 (8-2009)
Abstract
Abstract Background and Objectives Hematopoietic stem cells (HSCs) transplantation has been used for treating hematological disorders, immunodeficiencies, metabolic disorders and auto immune disease. In clinical application, the number of HSCs infused proved to be the major prognostic factor for engraftment and survival however, cord blood transplantation is limited in adults cause of their low number of cells. Consequently, we evaluated the potential of cell expansion and differentiation of cord blood cells in presence of different combination of cytokines. Materials and Methods In this interventional experimental study, umblical cord blood mononuclear cells (UCB MNC) were taken from apparently healthy mothers without any historical sign of diabetes during their pregnancy. We quantified and characterized an ex vivo expansion capacity of umbilical cord blood mononuclear cells (UCB MNC) in IMDM supplemented with 10% fetal bovine serum and various combination of SCF, TPO, Flt-3, GM-CSF and IL-3 as FSG3 T, FS3T, and FST for 3 weeks. Immunophenotyping and colony forming assay were performed at day 0, 7, 14 and 21 to characterize expanded cells in different groups. Results Total cell number was multiplied 5 times by all groups after 14 days of culture and the group with three cytokines of SCF, TPO, Flt-3 was shown to produce the highest percentage of hematopoitic stem cells and myeloid precursor cells among all groups during 2 weeks. Conclusions In this study, we showed an economical method for the expansion of umbilical cord blood stem cells with usage of UCB MNC and combination of SCF, TPO, and Flt-3. � Key words : Fetal blood, Hematopoietic Stem Cell, Cytokines, Interlukin-3, Granulocyte-macrophage Colony, Stimulating factor
M.h. Ahmadi, Kh. Etedali, Dr. M. Ebrahimi, S. Samimi, M. Mohammad, A. Khosh Akhlagh, Dr. T. Zandieh, Dr. M. Zarrabi,
Volume 6, Issue 4 (1-2010)
Abstract
Abstract Background and Objectives Umbilical cord blood has been used successfully as a source of hematopoietic stem cells for transplantation. Knowing that cord blood could be bacterially contaminated at the time it is being obtained and afterwards during the time it is being processed, we decided to perform the present study to determine any possible bacterial contamination and find out the root causes in Royan Cord Blood Bank (RCBB). Materials and Methods RCBB during 3 years received 3074 cord blood units (CBUs) upon which relevant investigation was made. In order to determine the root cause of the bacterial contamination, 800 CBUs were tested out of each of which two samples were taken. The data were analyzed with Chi-square by SPSS 16. Results Out of 3074 CBUs, 93 units (3.02%, CI 95%=2.3-3.6) were shown to have bacterial contamination. Out of the 800 units tested to detect the root cause, 26 samples (3.3%) were proved to be bacterial culture positive out of which 25 (96.2%) were shown to be contaminated at the time they were obtained and 1 (3.8%) during processing. The isolated bacteria were aerobic in 19 cases (73.1%) and anaerobic in 7 (26.9%). Conclusions The results show that bacterial contamination mostly is caused at the time blood is obtained from the cord in hospital obstetrics wards and most of the isolated bacteria were shown to be skin flora. Given the high value of cord blood stem cells and the risk of septic transplantation, it is necessary to prepare training programs for midwives and phlebotomists. Key words : Blood Banks, Cryopreservation, Umbilical Cord Blood
A. Raufi, A. Amini, M. Azadbakht, M. Aboozari, F. Fathi,
Volume 7, Issue 1 (3-2010)
Abstract
Abstract Background and Objectives Umbilical cord is one of the major sources of mesenchymal stem cells. In this study, the differentiative potential of human umbilical vein mesenchymal stem cells into hepatocyte-like cells has been investigated by cellular uptake of indocyanine green. Materials and Methods Human umbilical vein mesenchymal stem cells ( UVMSCs) were incubated for 2 weeks in the medium containing hepatocyte growth factor they were also treated with the medium containing oncostatin M for another 2 weeks. The differentiated cells were analyzed by uptake of indocyanine green (ICG), immunofluorescence analysis, and periodic acid-Schiff (PAS) staining. Results The differentiating cells showed transition from bipolar fibroblast-like morphology to round or oval shape. Cellular uptake of ICG was detected in differentiated cells. Glycogen storage was determined by PAS staining in differentiated cells which were immunoreactive to albumin. Conclusions Based on these observations, we can conclude that UVMSCs are able to differentiate into hepatocyte-like cells in vitro and ICG-staining is a useful marker to identify differentiated hepatocytes from human umbilical vein mesenchymal stem cells. � Keywords : Cell Differentiation, Umbilical Veins, Mesenchymal Stem Cells, Hepatocytes, Indocyanine Green�
A. R., Dr. A. Amini, Dr. M. Azadbakht, Dr. B. Nikkhoo, Dr. M. Aboozari, Dr. F. Fathi,
Volume 8, Issue 2 (5-2011)
Abstract
In vitro differentiation of human umbilical vein mesenchymal stem cells into hepatocyte-like cells Raufi A.1 , Amini A.1 , Azadbakht M.1 , Nikkhoo B.2,3 , Aboozari M.3 , Fathi F2,3 1 Razi University, Kermanshah, Iran 2 Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran 3 Kurdistan University of Medical Sciences, Sanandaj, Iran Abstract Background and Objectives Umbilical vein mesenchymal stem cells (UVMSC) which are multipotent precursors have been recently isolated. They are capable of differentiating into various cell types including neural and cardiac cells. In this study, the differentiation ability of human UVMSC into hepatocyte cells has investigated with concentration on hepatic genes expression profile. Materials and Methods The UVMSC were isolated and cultured in differentiation medium containing hepatocyte growth factor (HGF) and oncostatin M (OSM) using two step protocol. Differentiation of UVMSCs into cells expressing liver-specific genes was investigated by RT-PCR and immunocytochemistry. Results The cells showed the remarkable transition from bipolar fibroblast-like morphology to epithelial and polygonal shapes. The temporal gene expression pattern of hepatocyte-specific genes such as CK8, CK18, HNF3β, c-Met, AFP, TTR, G6P, Transthyretin, and ALB were detected during differentiation. The immunoflourescent analysis also showed that the differentiated cells were stained positively for CK-18 protein. Conclusions Findings indicate that UVMSC has a potential for differentiation into the hepatic like cells. Key words : Umbilical Veins, Mesenchymal Stem Cells, Hepatocyte Growth Factor, Oncostatin M Sci J Iran Blood Transfus Org 2011 8(2): 79-87 Received: 29 Nov 2010 Accepted: 15 Feb 2011
Correspondence: Fathi F., PhD of Anatomy. Associate Professor, Cellular and Molecular Research Center, Kurdistan University of Medical Sciences. P.O.Box: 66177-13446, Sanandaj, Iran. Tel: (+98871) 6661830 Fax : (+98871) 6660051 E-mail: 1- Biological safety cabinet |
1- Platelet Concentrate 2- Food and Drug Administration 3- Normal Skin Flora 4- Platelet Rich Plasma-Platelet Concentrate 5- Eosin-Methylene blue 6- Thioglycolate |
farfath@gmail.com
Dr. S. Amini, Dr. F. Fathi, Dr. K. Parivar, Dr. B. Nikkho, Dr. H. Sofimajidpour, Dr. J. Mobaleghi, Dr. B. Davari,
Volume 8, Issue 3 (8-2011)
Abstract
Abstract Background and Objectives Uncontrolled self renewal plays a direct function in different types of carcinoma progression. Here we examined the expression of self renewal regulatory factors such as Oct4, Nanog, Sox2, Nucleostemin, Zfx, Bmi-1 in colon, prostate, bladder and liver cancers in human samples and cancer cell lines. Materials and Methods We used RT-PCR to examine the expression of these genes in 10 tumors of bladder, 5 tumors of prostate, 5 tumors of colon , 5 normal tissues of colon, and cancer cell lines. The expression of Oct-4 and Nucleostemin at protein level was further determined by immunocytochemical (ICC) analysis in cancer cell lines. Results We designed specific primers to amplify a segment of Oct4, Nanog, Sox2, Nucleostemin, Bmi and Zfx. As expected DNA fragment of these genes based on designated primer was amplified in the PCR reaction. We detected the expression of these genes in almost all of the examined tumor samples and cancer cell lines that we used. Oct4 and Nucleostemin proteins were expressed in both nuclear and cytoplasmic in cancer cell lines. No immunoreactivity was observed in negative controls, which were incubated in the absence of primary antibody. Conclusions Collectively, our results indicated that in a tumor population a rare subpopulation of cells within the tumor cell mass has the potential of self renewal, and suggested that their expression can be used as potential tumor markers in diagnosis and/or prognosis of tumors. These results confirm the potential value of the cancer stem-cell theory in cancer therapy.
M. Ashki, Dr. N. Amirizadeh, Dr. M.a. Jalili, Dr. N. Hayati Roudbari, M.h. Mohammadi, M. Amani,
Volume 8, Issue 4 (1-2012)
Abstract
Abstract Background and Objectives During differentiation of mesenchymal stem cells (MSCs) into various cells, the expression of a variety of genes undergoes some changes in this study we decided to investigate the expression rate of some genes like osteopontin (OPN) and osteocalcin (OCN) during this process in order to find a better and faster way for these cells to be differentiated into osteoblasts . Material and Methods In this experimental study, the mononuclear cells of bone marrow were separated and then cultured in DMEM-LG culture media with 10% FBS. During some definite days, the RNA of differentiating cells was extracted. Then, the effective genes in osteogenesis like OPN and OCN were amplified by speciefic primers. The mesenchymal cells were cultured on 3D calcium phosphate scaffolds, and finally the activity rate of the alkaline phosphatase was examined . Results This research has demonstrated that in the process of differentiation, the expression of the two genes of OPN and OCN changed orderly with the maximum expression of OPN in the 6th day and the maximum expression of OCN in the 7th and 8th days of differentiation. The osteogenic differentiation of MSCs was not confirmed by the coloration of mineral sediments. The activity rate of alkaline phosphatase revealed the preference of 3D calcium phosphate scaffold to 2D environment in this differentiation. Conclusion The calcium phosphate scaffold positively affects the differentiation process. The expression of OPN and OCN genes changes during differentiation and can be used as away to a better and faster differentiation of these cells into osteoblast.
A. Dehghani Fard, N. Saki, M. Ahmadvand, M. Mahmoodinia Maymand, M. Mosahebi Mohammadi, Dr. M. Soleimani,
Volume 8, Issue 4 (1-2012)
Abstract
Abstract Background and Objectives Mesenchymal Stem Cells have extensive potential to proliferate and differentiate into different cell lineages. Their differentiation capability in vivo and in vitro makes them ideal tools for tissue engineering and regenerative medicine. Materials and Methods In the present study more than 100 recent published articles which are about isolation, culture and differentiation of MSCs were reviewed for application of MSC in regenerative medicine and tissue engineering. Results Clinical applications of MSCs seem to be in two distinctive lines: bio-scaffold design without immunological responses as well as multipotent stem cell without clinical obstacles. Conclusions MSCs due to their capacity of self-renewability, multilineage differentiation and immune modulatory effects are of great therapeutic potential for cell and gene therapy of congenital and degenerative disorders.
M. Mohammadzadeh, R. Halabian, M. Mohammadipoor, A.a. Kiani, Dr. A. Gharehbaghian, Dr. N. Amirizadeh, Dr. M. Habibi Roudkenar,
Volume 8, Issue 4 (1-2012)
Abstract
Abstract Background and Objectives Poor viability of Mesenchymal Stem Cells (MSCs) following transplantation is one of the major challenges in their therapeutic application. Manipulation of MSCs by the genetic engineering method is one of the strategies used to protect the cells against cytotoxic microenvironment. However, maintaining multi differentiation capacity of MSCs following manipulation is important. We investigated if the manipulation of MSCs with NRF2 affects the multi differentiation capacity. Materials and Methods MSCs were isolated from bone marrow. NRF2 was isolated and TOPO cloned into the pENTR vector. The recombinant vector was transferred into pAD/CMV/V5-DEST vector by gateway technology. Recombinant adenovirus was produced in AD293 cells, followed by being infected into MSCs. Expression of NRF2 was verified by RT-PCR. The NRF2 engineered MSCs were exposed to stress conditions followed by the evaluation of the cells viability and apoptosis. Finally, NRF2 expressing MSCs differentiation into osteoblast and adipocyte lineages was studied. Results NRF2 was successfully expressed in MSCs. NRF2- MSCs differentiation into osteoblast and adipocyte lineages indicating overexpression of NRF2 does not affect the differentiation property of MSCs. Conclusions Expression of NRF2, a well known cytoprotective factor, by using adenovirus expression system does not intervene in the differentiation capacity of MSCs. NRF2-MSCs might be applicable for stem cell-based cell therapy in future.
Dr. S. Rahgozar, M. Entezar-E-Ghaem, M. Abedi, F. Montazeri,
Volume 9, Issue 3 (8-2012)
Abstract
Abstract Background and Objectives Hemoglobinopathies are heterogenic hereditary disorders in which mutations lead to abnormal production of hemoglobin chains, or change in the normal sequence of the hemoglobin amino acids. Thalassemia and sickle cell anemia (SCA) are the most prevalent hemoglobinopathies. Patients with the above mentioned disorders often require hypertransfusion regimens. However, continued blood transfusion may contribute to iron overload and consequent organ deterioration, in addition to viral infections. The aim of this review article is to evaluate recent treatment programs regarding these disorders. Materials and Methods This paper is evaluating the newest protocols applied for the treatment of thalassemia and SCA by reviewing 51 references using Ebsco, Elsevier, Pubmed and OVID databases. Results Blood transfusion and medical treatments may improve the lifestyle or increase the lifespan of patients with hemoglobinopathies. However, hematopoietic stem cell transplantation (HSCT) is presented as the only curative therapy for thalassemia and SCA. On the other hand, HSCT may contribute to complications such as infertility and gonadal failure, especially in women, chronic graft-versus-host disease (GVHD), and potential secondary malignancies. Conclusions In spite of the limitations and complications attributed to HSCT, this method is proved to be the most effective way for treatment of hemoglobinopathies. Different cells and novel strategies used to modify transplantation are introduced in this article.
R. Ranjbaran, Dr. H. Abolghasemi, N. Nasiri, A. Oodi, Dr. M. Nikougoftar , Dr. N. Amirizadeh,
Volume 9, Issue 3 (8-2012)
Abstract
Abstract Background and Objectives Stem cell transplantation has achieved high success for treatment but the use of this source has some limitation. Cord blood transplantation is clinically limited by its low progenitor cell contents such as CD34+. The aim of this study was to evaluate the effect of mesenchymal stem cells (MSCs) on replication of CD34+ in cord blood. Materials and Methods In this experimental study MSCs were isolated and cultured from BM mononuclear cells. MSCs were used for co-culture in two systems of 2D and 3D cultures. After isolation, umbilical cord blood CD34+ cells were cultured in two different systems. The level of expansion in these systems was evaluated by cell count, analysis of CD34+ expression by flowcytometery, and CFU assay . Results Expansion of cord blood HSCs was evaluated at three different culture systems at three different times (day3, day 7 and day 10): the medium with cytokine, 2D medium with cytokine + MSCs, and 3D medium with cytokine + MSCs on scaffold. The fold increase of TNC, CFU, and CD34+ cells at day 10 for the former medium was 87 ± 6, 84 ± 8, 11 ± 3, for the second 103 ± 8, 111 ± 9, 14 ± 3, and for the latter 127 ± 10, 170 ± 14, 20 ± 4, respectively. The highest expansion was observed in the 3D co-culture with MSCs after 10 days. Conclusions The results have demonstrated that the co-culture of CB HSCs with mesenchymal cells could increase the rate of expansion especially in the 3D system.
, , , , , , ,
Volume 9, Issue 3 (8-2012)
Abstract
Abstract Background and Objectives Micro RNAs are a class of small non-coding RNAs which have been recently shown to play a crucial role in major cellular processes such as development and differentiation through post-transcriptional regulation. The role of these epigenetic elements has been also demonstrated in hematopoietic lineage differentiation and there is a large body of evidence that miR-150 and miR-146a are responsible for T lymphocyte differentiation. Our goal was to examine the effect of miR-150 and miR-146a overexpression in hematopoietic stem cells and its ability to differentiate these cells into a T lymphoid cell. Materials and Methods In this experimental study CD133+ stem cells were employed as hematopoietic stem cells and permanent overexpression of miR-150 and miR-146a was established. The differentiation progress was tracked by flowcytometry for lymphocyte markers such as CD4 and CD8. Moreover, expression of miR-146a and miR-150 by RT-PCR and quantitative real time PCR after 7 days was studied. Results The cells showed T lymphoid characteristics 7 days after transduction. CD4 and especially CD8 expression significantly increased. Conclusions In conclusion, miR-150 and miR-146a overexpression is an effective factor in T cytotoxic differentiation and they have the ability of directing CD133+ hematopoietic stem cells to express T lymphoid characteristics.
Z.s. Hashemi, Dr. M. Forouzandeh Moghadam, Dr. M. Soleimani, Dr. Gh. Khamisipour, M. Mossahebi Mohammadi,
Volume 9, Issue 3 (8-2012)
Abstract
Abstract Background and Objectives Bone marrow transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming by minimum manipulation, mineralized bone allograft (MBA) scaffold coated by UCB-USSC (unrestricted somatic stem cells) was used to expand CD34+ cells from UCB. Materials and Methods In this experimental study, UCB-USSCs were isolated and characterized by morphologic and immunophenotypical analysis. Then they were seeded on MBA (for 3D figuration) and culture plate (for 2D figuration) as a feeder layer. CD34+ cells were isolated from the UCB (by MACS method) and were expanded in 2D and 3D conditions for 3 weeks. At the end, cell count, flow cytometry, cologenic assay, and LTC-IC were done. Results After 3 weeks, ex vivo expansion of UCB-CD34+ was enhanced 250 ± 13.2 in 3D. The highest CFC expansion and LTC-IC were observed at day 14. Flow cytometry analysis showed the lowest percentage in 3D culture. Conclusions USSCs by 3D MBA structure produce the hematopoietic cytokine, extracellular matrix and binding molecules they also create the intercellular interaction that can be used as a suitable feeder layer. It provides an ex vivo mimicry of bone marrow niche by enhancing surface/volume ratio. This model could be a suitable and alternative environment for HSCs expansion and hematopoiesis.
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